85 BACTERIAL ENDOTOXINS TEST

Transcription

1 Offiil April 1, Bteril Enotoxins Test 1 85 BACTERIAL ENDOTOXINS TEST Portions of this generl hpter hve een hrmonize with the orresponing texts of the Europen Phrmopoei n/or the Jpnese Phrmopoei. Those portions tht re not hrmonize re mrke with symols ( ) to speify this ft. The Bteril Enotoxins Test (BET) is test to etet or quntify enotoxins from Grm-negtive teri using moeoyte lyste from the horseshoe r (Limulus polyphemus or Thypleus trienttus). There re three tehniques for this test: the gel-lot tehnique, whih is se on gel formtion; the turiimetri tehnique, se on the evelopment of turiity fter levge of n enogenous sustrte; n the hromogeni tehnique, se on the evelopment of olor fter levge of syntheti peptie-hromogen omplex. Proee y ny of the three tehniques for the test. In the event of out or ispute, the finl eision is me se upon the gel-lot tehnique unless otherwise inite in the monogrph for the prout eing teste. The test is rrie out in mnner tht vois enotoxin ontmintion. APPARATUS Depyrogente ll glsswre n other het-stle mterils in hot ir oven using vlite proess. 1 A ommonly use minimum time n temperture is 30 minutes t 250. If employing plsti pprtus, suh s miropltes n pipet tips for utomti pipetters, use pprtus tht is shown to e free of etetle enotoxin n oes not interfere in the test. [NOTE In this hpter, the term tue inlues ny other reeptle suh s mirotiter well.] PREPARATION OF SOLUTIONS Stnr Enotoxin Stok Solution A Stnr Enotoxin Stok Solution is prepre from USP Enotoxin Referene Stnr tht hs een lirte to the urrent WHO Interntionl Stnr for Enotoxin. Follow the speifitions in the pkge leflet n on the lel for preprtion n storge of the Stnr Enotoxin Stok Solution. Eno- toxin is expresse in Enotoxin Units (EU). [NOTE One USP Enotoxin Unit (EU) is equl to one Interntionl Unit (IU) of enotoxin.] Stnr Enotoxin Solutions After mixing the Stn- r Enotoxin Stok Solution vigorously, prepre pproprite seril ilutions of Stnr Enotoxin Solution, using Wter for BET. Use ilutions s soon s possile to voi loss of tivity y sorption. Smple Solutions Prepre the Smple Solutions y is- solving or iluting rugs, or tking wshes from meil evies using Wter for BET. Some sustnes or preprtions my e more ppropritely issolve, ilute, or ex- trte in other queous solutions. If neessry, just the ph of the solution to e exmine (or ilution thereof) so tht the ph of the mixture of the lyste n Smple Solution flls within the ph rnge speifie y the lyste mnuf- turer, usully 6.0 to 8.0. The ph my e juste y use of n i, se, or suitle uffer s reommene y the lyste mnufturer. Ais n ses my e prepre from onentrtes or solis with Wter for BET in ontiners free of etetle enotoxin. Buffers must e vlite to e free of etetle enotoxin n interfering ftors. Chnge to re: DETERMINATION OF MAXIMUM VALID DILUTION (MVD) REAGENTS AND TEST SOLUTIONS The mximum vli ilution is the mximum llowle ilution of speimen t whih the enotoxin limit n e etermine. Determine the MVD from the following Amoeoyte Lyste A lyophilize prout otine eqution: from the lyste of moeoytes (white loo ells) from the horseshoe r (Limulus polyphemus or Thypleus MVD = (Enotoxin Limit Conentrtion of Smple trienttus). This regent refers only to prout mnuf- Solution)/(λ) ture in orne with the regultions of the ompetent uthority. [NOTE Amoeoyte Lyste rets to some β-glu- Enotoxin Limit The enotoxin limit for prenterl ns in ition to enotoxins. Amoeoyte Lyste preprrugs, efine on the sis of ose, equls K/M 2, where K tions tht o not ret to gluns re ville: they re is threshol pyrogeni ose of enotoxin per kg of oy prepre y removing the G ftor reting to gluns from weight, n M is equl to the mximum reommene o- Amoeoyte Lyste or y inhiiting the G ftor reting syslus ose of prout per kg of oy weight. When the protem of Amoeoyte Lyste n my e use for enotoxin ut is to e injete t frequent intervls or infuse ontinutesting in the presene of gluns.] ously, M is the mximum totl ose ministere in single Wter for Bteril Enotoxins Test (BET) Use Wter hour perio. The enotoxin limit for prenterl rugs is for Injetion or wter proue y other proeures tht speifie in the iniviul monogrph in units suh s EU/ shows no retion with the lyste employe, t the ete- ml, EU/mg, EU/Unit of iologil tivity, et. tion limit of the regent. Conentrtion of Smple Solution Lyste TS Dissolve Amoeoyte Lyste in Wter for BET, mg/ml: in the se of enotoxin limit speifie y weight or in uffer reommene y the lyste mnufturer, y (EU/mg); gentle stirring. Store the reonstitute lyste, refrigerte or Units/mL: in the se of enotoxin limit speifie y unit frozen, oring to the speifitions of the mnufturer. of iologil tivity (EU/Unit); 1For vliity test of the proeure for intivting enotoxins, see Dry-Het 2 K is 5 USP-EU/kg for ny route of ministrtion other thn intrthel (for Steriliztion uner Steriliztion n Sterility Assurne of Compenil Artiles whih K is 0.2 USP-EU/kg oy weight). For riophrmeutil prouts Use Lyste TS hving sensitivity of not less thn 0.15 Enotoxin Unit not ministere intrthelly, the enotoxin limit is lulte s 175/V, per ml. where V is the mximum reommene ose in ml. For intrthelly ministere riophrmeutils, the enotoxin limit is otine y the formul 14/V. For formultions (usully ntiner prouts) ministere on per squre meter of oy surfe, the formul is K/M, where K = 2.5 USP-EU/ kg (IRA 1-Apr-2011) n M is the (mximum ose/m2 /hour 1.80 m 2 )/70 Kg.

2 2 85 Bteril Enotoxins Test Offiil April 1, 2011 Tle 1. Preprtion of Solutions for the Inhiition/Enhnement Test for Gel-Clot Tehniques Enotoxin Conentrtion/ Solution to Whih Enotoxin Dilution Enotoxin Numer of Solution Is Ae Diluent Ftor Conentrtion Replites A None/Smple Solution 4 B 2λ/Smple Solution Smple Solution 1 2λ 4 2 1λ λ λ 4 C 2λ/Wter for BET Wter for BET 1 2λ 2 2 1λ λ λ 2 D None/Wter for BET 2 Solution A: A Smple Solution of the preprtion uner test tht is free of etetle enotoxins. Solution B: Test for interferene. Solution C: Control for lele lyste sensitivity. Solution D: Negtive ontrol of Wter for BET. ml/ml: when the enotoxin limit is speifie y volume positive. A result is negtive if n intt gel is not forme. (EU/mL). The test is onsiere vli when the lowest onentrtion λ: the lele sensitivity in the Gel-Clot Tehnique (EU/mL) of the stnr solutions shows negtive result in ll replior the lowest onentrtion use in the stnr regression te tests. urve for the Turiimetri Tehnique or Chromogeni The enpoint is the smllest onentrtion in the series of Tehnique. eresing onentrtions of stnr enotoxin tht lots the lyste. Determine the geometri men enpoint y lulting Chnge to re: the men of the logrithms of the enpoint on- entrtions of the four replite series n then tking the ntilogrithm of the men vlue, s inite in the following formul: GEL-CLOT TECHNIQUE Preprtory Testing Test for Confirmtion of Lele Lyste Sensitivity Confirm in four replites the lele sensitivity, λ, ex- presse in EU/mL of the lyste prior to use in the test. The test for onfirmtion of lyste sensitivity is to e rrie out when new th of lyste is use or when there is ny hnge in the test onitions tht my ffet the outome of the test. Prepre stnr solutions hving t lest four onentrtions equivlent to 2λ, λ, 0.5λ, n 0.25λ y iluting the USP Enotoxin RS with Wter for BET. Mix volume of the Lyste TS with n equl volume (suh s 0.1-mL liquots) of one of the Stnr Enotoxin Solutions in eh test tue. When single test vils or mpuls ontining lyophilize lyste re use, solutions iretly to the vil or mpul. Inute the retion mixture for onstnt perio oring to the iretions of the lyste mnufturer (usully t 37 ± 1 for 60 ± 2 minutes), voi- ing virtion. To test the integrity of the gel, tke eh tue in turn iretly from the inutor n invert it through out 180 in one smooth motion. If firm gel hs forme tht remins in ple upon inversion, reor the result s Geometri Men Enpoint Conentrtion = ntilog (Σe/f) The gel-lot tehnique is for eteting or quntifying en- otoxins se on lotting of the lyste regent in the pres- ene of enotoxin. The minimum onentrtion of eno- toxin require to use the lyste to lot uner stnr onitions is the lele sensitivity of the lyste regent. To ensure oth the preision n vliity of the test, perform the tests for onfirming the lele lyste sensitivity n for interfering ftors s esrie uner Preprtory Testing, immeitely elow. where Σe is the sum of the log enpoint onentrtions of the ilution series use, n f is the numer of replite test tues. The geometri men enpoint onentrtion is the mesure sensitivity of the lyste (in EU/mL). If this is not less thn 0.5λ n not more thn 2λ, the lele sensitivity is onfirme n is use in tests performe with this lyste. Test for Interfering Ftors Usully prepre solutions (A D) s shown in Tle 1, n perform the inhiition/en- hnement test on the Smple Solutions t ilution less thn the MVD, not ontining ny etetle enotoxins, operting s esrie for Test for Confirmtion of Lele Lyste Sensitivity. The geometri men enpoint onentrtions of Solutions B n C re etermine using the formul esrie in the Test for Confirmtion of Lele Lyste Sensitivity. The test for interfering ftors must e repete when ny onition hnges tht is likely to influene the result of the test. (IRA 1-Apr-2011) The test is onsiere vli when ll replites of Solu- tions A n D show no retion n the result of Solution C onfirms the lele sensitivity. If the sensitivity of the lyste etermine in the presene of Solution B is not less thn 0.5λ n not greter thn 2λ, the Smple Solution oes not ontin ftors tht interfere uner the experimentl onitions use. Otherwise, the Smple Solution to e exmine interferes with the test. If the smple uner test oes not omply with the test t ilution less thn the MVD, repet the test using greter ilution, not exeeing the MVD. The use of more sensitive lyste permits greter ilution of the smple to e exmine, n this my ontriute to the elimintion of interferene. Interferene my e overome y suitle tretment, suh s filtrtion, neutrliztion, ilysis, or heting. To estlish

3 Offiil April 1, Bteril Enotoxins Test 3 Tle 3. Preprtion of Solutions for the Gel-Clot Assy Enotoxin Conentrtion/ Solution to Whih Enotoxin Dilution Enotoxin Numer of Solution Is Ae Diluent Ftor Conentrtion Replites A None/Smple Solution Wter for BET B 2λ/Smple Solution 1 2λ 2 C 2λ/Wter for BET Wter for BET 1 2λ 2 2 1λ λ λ 2 D None/Wter for BET 2 Solution A: Smple Solution uner test t the ilution, not to exee the MVD, with whih the Test for Interfering Ftors ws omplete. Susequent ilution of the Smple Solution must not exee the MVD. Use Wter for BET to mke ilution series of four tues ontining the Smple Solution uner test t onentrtions of 1, 1 /2, 1 /4, n 1 /8 reltive to the onentrtion use in the Test for Interfering Ftors. Other ilutions up to the MVD my e use s pproprite. Solution B: Solution A ontining stnr enotoxin t onentrtion of 2λ (positive prout ontrol). Solution C: Two replites of four tues of Wter for BET ontining the stnr enotoxin t onentrtion of 2λ, λ, 0.5λ, n 0.25λ, respetively. Solution D: Wter for BET (negtive ontrol). plies with the test if negtive result is foun for oth repli- tes of Solution A. The preprtion oes not omply with the test if positive result is foun for one or oth replites of Solution A. However, if the preprtion oes not omply with the test t ilution less thn the MVD, the test my e repete using greter ilution, not exeeing the MVD. tht the hosen tretment effetively elimintes interferene without loss of enotoxins, perform the ssy esrie ove using the preprtion to e exmine to whih USP Enotoxin RS hs een e n whih hs then een sumitte to the hosen tretment. Limit Test Quntittive Test Proeure Prepre Solutions A, B, C, n D s shown in Tle 2, n perform the test on these solutions following the proeure for Test for Confirmtion of Lele Lyste Sen- Proeure The test quntifies teril enotoxins in sitivity uner Preprtory Testing, ove. Smple Solutions y titrtion to n enpoint. Prepre Solutions A, B, C, n D s shown in Tle 3, n test these solutions y following the proeure in the Test for Confir- Tle 2. Preprtion of Solutions for the Gel-Clot Limit Test mtion of Lele Lyste Sensitivity uner Preprtory Testing. Enotoxin Conentrtion/ Clultion n Interprettion The test is onsiere Solution to Whih Numer of vli when the following three onitions re met: (1) Both Solution replites of negtive ontrol Solution D re negtive; (2) Enotoxin Is Ae Replites A None/Dilute Smple Solution 2 Both replites of positive prout ontrol Solution B re positive; n (3) The geometri men enpoint onentr- B 2λ/Dilute Smple Solution 2 tion of Solution C is in the rnge of 0.5λ to 2λ. C 2λ/Wter for BET 2 To etermine the enotoxin onentrtion of Solution A, D None/Wter for BET 2 lulte the enpoint onentrtion for eh replite y * Prepre Solution A n the positive prout ontrol Solution B using multiplying eh enpoint ilution ftor y λ. The enoilution not greter thn the MVD n tretments s for the Test for toxin onentrtion in the Smple Solution is the enpoint Interfering Ftors uner Preprtory Testing. The positive ontrol Solutions B n C ontin the Stnr Enotoxin Solution t onentrtion orre ilute Smple Solution, lulte the onentrtion of en- onentrtion of the replites. If the test is onute with sponing to twie the lele lyste sensitivity. The negtive ontrol Solution D onsists of Wter for BET. otoxin in the originl Smple Solution y multiplying y the ilution ftor. If none of the ilutions of the Smple Solution is positive in vli ssy, report the enotoxin onen- Interprettion The test is onsiere vli when oth replites of Solution B n C re positive n those of Soluport s less thn λ times the lowest ilution ftor of the trtion s less thn λ (if the ilute smple ws teste, re- tion D re negtive. When negtive result is foun for oth replites of Solution A, the preprtion uner test smple.) If ll ilutions re positive, the enotoxin onen- omplies with the test. When positive result is foun for trtion is reporte s equl to or greter thn the gretest oth replites of Solution A, the preprtion uner test ilution ftor multiplie y λ (e.g., initil ilution ftor oes not omply with the test. times 8 times λ in Tle 3). When positive result is foun for one replite of Solutest if the onentrtion of enotoxin in oth replites is The preprtion uner test meets the requirements of the tion A n negtive result is foun for the other, repet the test. In the repet test, the preprtion uner test om- less thn tht speifie in the iniviul monogrph.

4 4 85 Bteril Enotoxins Test Offiil April 1, 2011 Tle 4. Preprtion of Solutions for the Inhiition/Enhnement Test for Photometri Tehniques Solution to Whih Solution Enotoxin Conentrtion Enotoxin Is Ae Numer of Replites A None Smple Solution Not less thn 2 B Mile onentrtion of the stnr urve Smple Solution Not less thn 2 C At lest 3 onentrtions (lowest onentrtion is Wter for BET Eh not less thn 2 esignte λ) D None Wter for BET Not less thn 2 Solution A: The Smple Solution my e ilute not to exee MVD. Solution B: The preprtion uner test t the sme ilution s Solution A, ontining e enotoxin t onentrtion equl to or ner the mile of the stnr urve. Solution C: The stnr enotoxin t the onentrtions use in the vlition of the metho esrie for Assurne of Criteri for the Stnr Curve uner Preprtory Testing (positive ontrols). Solution D: Wter for BET (negtive ontrol). PHOTOMETRIC QUANTITATIVE TECHNIQUES onentrtions within the rnge inite y the lyste mnufturer to generte the stnr urve. Perform the ssy using t lest three replites of eh stnr enotoxin onentrtion oring to the mnufturer s instru- Turiimetri Tehnique tions for the lyste (volume rtios, inution time, temperture, ph, et.). If the esire rnge is greter thn two logs This tehnique is photometri ssy mesuring inreses in the kineti methos, itionl stnrs shoul e inin retnt turiity. On the sis of the prtiulr ssy lue to rket eh log inrese in the rnge of the stnpriniple employe, this tehnique my e lssifie s ei- r urve. The solute vlue of the orreltion oeffither n enpoint-turiimetri ssy or kineti-turiimet- ient, r, must e greter thn or equl to 0.980, for the ri ssy. The enpoint-turiimetri ssy is se on the rnge of enotoxin onentrtions set up. quntittive reltionship etween the onentrtion of en- Test for Interfering Ftors Selet n enotoxin onotoxins n the turiity (sorne or trnsmission) of entrtion t or ner the mile of the enotoxin stnr the retion mixture t the en of n inution perio. urve. Prepre Solutions A, B, C, n D s shown in Tle 4. The kineti-turiimetri ssy is metho to mesure ei- Perform the test on Solutions A, B, C, n D t lest in uplither the time (onset time) neee to reh preetermine te oring to the instrutions for the lyste employe, sorne or trnsmission of the retion mixture, or the for exmple, onerning volume of Smple Solution n Lyrte of turiity evelopment. The test is rrie out t the ste TS, volume rtio of Smple Solution to Lyste TS, inuinution temperture reommene y the lyste mnu- tion time, et. fturer (whih is usully 37 ± 1 ). The test is onsiere vli when the following onitions re met. 1. The solute vlue of the orreltion oeffiient of the Chromogeni Tehnique stnr urve generte using Solution C is greter thn or equl to This tehnique is n ssy to mesure the hromophore 2. The result with Solution D oes not exee the limit of relese from suitle hromogeni peptie y the rethe lnk vlue require in the esription of the lyste retion of enotoxins with lyste. On the sis of the prtiulr gent employe, or it is less thn the enotoxin etetion ssy priniple employe, this tehnique my e lssifie s limit of the lyste regent employe. either n enpoint-hromogeni ssy or kineti-hromo- Clulte the men reovery of the e enotoxin y geni ssy. The enpoint-hromogeni ssy is se on sutrting the men enotoxin onentrtion in the soluthe quntittive reltionship etween the onentrtion of tion, if ny (Solution A, Tle 4), from tht ontining the enotoxins n the relese of hromophore t the en of e enotoxin (Solution B, Tle 4). In orer to e onsin inution perio. The kineti-hromogeni ssy is ere free of ftors tht interfere with the ssy uner the metho to mesure either the time (onset time) neee to onitions of the test, the mesure onentrtion of the reh preetermine sorne of the retion mixture, enotoxin e to the Smple Solution must e within or the rte of olor evelopment. The test is rrie out t 50% to 200% of the known e enotoxin onentrtion the inution temperture reommene y the lyste fter sutrtion of ny enotoxin etete in the solution mnufturer (whih is usully 37 ± 1 ). without e enotoxin. When the enotoxin reovery is out of the speifie Preprtory Testing rnge, the Smple Solution uner test is onsiere to ontin interfering ftors. Then, repet the test using greter To ssure the preision or vliity of the turiimetri n ilution, not exeeing the MVD. Furthermore, interferene hromogeni tehniques, preprtory tests re onute to of the Smple Solution or ilute Smple Solution not to ex- verify tht the riteri for the stnr urve re vli n ee the MVD my e eliminte y suitle vlite tht the smple solution oes not interfere with the test. tretment, suh s filtrtion, neutrliztion, ilysis, or het Vlition for the test metho is require when onitions tretment.to estlish tht the tretment hosen effetively tht re likely to influene the test result hnge. elimintes interferene without loss of enotoxins, perform the ssy esrie ove using the preprtion to e ex- Assurne of Criteri for the Stnr Curve The test mine to whih Stnr Enotoxin hs een e n must e rrie out for eh lot of lyste regent. Using the whih hs then een sumitte to the hosen tretment. Stnr Enotoxin Solution, prepre t lest three enotoxin

5 Offiil April 1, Bteril Enotoxins Test 5 Test Proeure Clultion In photometri ssys, the preprtion uner test om- plies with the test if the men enotoxin onentrtion of the replites of Solution A, fter orretion for ilution n onentrtion, is less thn the enotoxin limit for the prout. Clulte the enotoxin onentrtion of eh of the replites of Solution A using the stnr urve generte y the positive ontrol Solution C. The test is onsiere vli when the following three requirements re met. 1. The results of the ontrol Solution C omply with the requirements for vlition efine for Assurne of Criteri for the Stnr Curve uner Preprtory Testing. 2. The enotoxin reovery, lulte from the onentrtion foun in Solution B fter sutrting the onentrtion Follow the proeure esrie for Test for Interfering Ftors uner Preprtory Testing, immeitely ove. of enotoxin foun in Solution A, is within the rnge of 50% to 200%. 3. The result of the negtive ontrol Solution D oes not exee the limit of the lnk vlue require in the esrip- tion of the lyste employe, or it is less thn the enotoxin etetion limit of the lyste regent employe. Interprettion